| [Background]Different from the central nerve system, the peripheral nerve has a strong potential for regeneration; when an appropriate microenvironment is provided, the regenerating axons extend their processes into the distal Bungner bands to achieve regeneration and rehabilitate normal function. But the function of peripheral nerve injury is still difficult to get satisfactory recovery. Epineurium neurorrhaphy to recover nerve continuity was the traditional choice of peripheral nerve mutilation without sufficient nerve defects, whereas this technique is put out of its power to ensure exact coaptation of the millions of nerve ends. Therefore dissatisfactory rehabilitation after the traditional neurorrhaphy is predictable. However, in the early 1900s, Cajal pioneered the concept that axons regenerate from neurons and are guided by chemotrophic substances, which provided new ideas for the treatment of nerve injury. Owing to the nerve selective regeneration theory, which has been repeated and confirmed, a novel neurorrhaphy method was advanced to provide an appropriate gap between the distal and proximal severed stumps to facilitate nerve spontaneous selection when it regenerates across this space. This small gap sleeve bridging peripheral nerve method has obtained good effects in an animal experiment study and in clinical practice, and might take the place of epineurium neurorrhaphy to be the recommended method to repair peripheral nerve injury. However, the best gap distance of small gap sleeve bridging peripheral nerve method in nerve repair is inconclusive. If the distance of the gap is too long, the proximal stump of the regenerating nerve is difficult to extend through the gap to the distal stump, if the distance is too short, and the potential role of selective regeneration of nerve can not fully play. Therefore, it’s of great significance to explore the optimal gap distance of small gaps suture on the surgical treatment of peripheral nerve injury.[Objective]To investigate the effects of repairing peripheral nerves with different lengths of small gap suture, this experiment using poly lactic acid membrane small gap suture repair of rat common peroneal nerve injury and to explore the effects of different clearances suture repair of peripheral nerve injury.[Methods]1. Animal model establishment:40 SPF Sprague-Dawley (SD) rats from the experimental animal center of Southern Medical University, weight 250-300g, aged 3 months, quality certificate number 44002100004044, were fed in the animal feeding room(temperature 23±2 C, humidity 50±10%, daily light 12h, ventilation for 8 to 12 hours) for 1week to adapt to the new environment.2 rats were fed freely in each cage. Rats were kept from food and water for 12 hours before operation, with preoperative intramuscular injection of penicillin sodium (200000U/kg) for prevention of postoperative infection and intraperitoneal injection of 0.1% sodium O.lml/100g for intraperitoneal injection anesthesia. After anesthesia, the hair of the right lower limbs and the waist and belly was cleared away with an electric shaver knife. Rats were fixed on the operation board in prone position. Then disinfected the operation area with iodophor. The experimental animals were randomly divided into 4 groups(10 rats in each group) according to the operation method and the gap distance between the nerve stumps and:In strict accordance with the principles of aseptic, from the right posterolateral incision, we cut apart the skin, fascia, and separated the muscles bluntly. Pulling the muscle to both sides, under the microscope observation, expose and gently and bluntly separated of the common peroneal nerve. The common peroneal nerve was cut at 5mm proximal from the muscular gate by sharp microscopic tissue sissors. According to the shape of the common peroneal nerve stumps and the nutrient vessels, the two ends of nerve stumps were coaptated together in accurate alignment. Rectangular polylactic acid membrane clipping about 5mmx4mm, will be placed between the nerve stumps, while keeping 1 mm,2 mm,3mm, Omm(referred to as Group E1, Group E2, Group E3, Group N) of gap between the nerve stumps, respectively. The epineurium of distal and proximal ends of nerve stumps were stitched to the polylactic acid membrane on both sides. Curled the polylactic acid membrane to surround the nerve stumps to form a tube which matched the diameter of the nerve stumps. Finally, the free edge of the poly lactic acid membrane was sutured. Used saline to wash the operative field, with complete hemostasis. Sutured the skin and fascia.2.observation index:①Observe recovery effect of the ankle and toe dorsiflexion of experimental rats after operation;②Nerve electrophysiological examination:Anesthetized the rats through intraperitoneal injection of 0.1% sodium O.lml/100g. After anesthesia, the hair of the right lower limbs and the waist and belly was cleared away with an electric shaver knife. Rats were fixed on the operation board in prone position. Measured the nerve conduction velocity of the right peroneal nerve by electromyography.③6 weeks after the surgery, the right common peroneal nerve nerves were exposed under microscope for anatomy observation.④Tissue morphology observation:The suture site and the small gap and the distal 5mm of regenerated nerves were separated and cut. The fixed regenerated nerves were fixed by 4% formalin, embedded by paraffin, and sliced in longitudinal, transverse row. Routine hematoxylin eosin staining (HE staining) was performed. Immunohistochemical reaction by Anti-S100 antibody-Astrocyte and Anti-200kD Neurofilament Heavy was performed. Tissue morphology of regenerated nerves was observed. Plus Image-Pro 6 image analysis software was used to measure the number of nerve fibers.⑤Ultrastructure of regenerative nerve observation:the distal lmm of regenerated nerves were separated and cut and fixed by glutaraldehyde, stained with uranyl acetate and lead citrate. Observation of ultrastructure of nerve regeneration was performed throgh transmission electron microscope.⑥Measurement of muscle wet weight ratio:Bilateral anterior lateral muscle groups of the lower leg in rats were completly isolated and cut off. Blood on the surface of muscles was wiped away with dry gauze. The wet weight of muscles was measured by electronic balance. The muscle wet weight ratio was calculated. Wet weight ratio= the wet weight of the anterior lateral leg of the surgical side/the wet weight of the anterior lateral leg of the normal side.3. Statistical analysisThe data were expressed as the mean±SD, and analyzed using SPSS 20.0 software. The differences at different time points were compared using one-way analysis of variance and independent-sample t-test. A value of P< 0.05 was considered statistically significant.[Result]1.The ankle and toe dorsiflexion recovery in rats after operation:After the operation, the lateral anterior muscles of the right leg were paralyzed, the right ankles and toes failed to dorsiflex. It took 25 days for group N to regain toe and ankle joint dorsal flexion postoperatively, while 16,22 and 30 days for group El, E2, E3 respectively.2.Nerve electrophysiological examination (common nerve conduction velocity): At the 6th week after operations, the right side of the common peroneal nerve conduction velocity showed:Group El> Group E2> Group N> Group E3, the difference was statistically significant (P< 0.05).3.Nerve morphology under the microscope:At the 6th week after operations, the right peroneal nerves were exposed, and the morphology of the regenerated nerves were observed under the microscope. Poly lactic acid membrane has been completely degraded, no nerves and surrounding tissues adhesion. Observed through the microscope, the regenerated nerves of Group El were smooth and the diameters of the nerves were basically normal. In Group E2, the regenerated nerves were not smooth, and the diameters of the nerves were slightly smaller. In Group E3, the regenerated nerves were significantly expanded, and the distal parts of nerves were obviously thinner. In Group N, the regenerated nerves were expanded, and the distal parts of nerves were thin.4.Tissue morphology observation:After HE staining, tissue morphology under the microscope showed:In the 1mm small suture group (Group E1), the regenerated nerve fibers were arranged in a neat and orderly manner, with a large number of nerve fibers and a large number of Schwann cells, and the myelin sheath was clearly visible, with no connective tissue proliferation. In the 2mm small suture group (Group E2), the arrangement of the regenerated nerve fibers was less orderly, and the nerve fibers were thinner, and the number of Schwann cells was less, and the myelin sheath was less clear, and the proliferation of connective tissue was found. In the 3mm small suture group (Group E3), the regenerative nerve showed expansive growth, curled into a group, arranged in disorder, and the number of nerve regeneration was the least, and the diameter of the smallest, with blurred myelin, more connective tissue hyperplasia. In the Omm gapless suture group (Group N), the regenerated nerve fibers is disordered, with small diameter of the nerve, less Schwann cells, and the myelin sheath is not obvious.Anti-S100 antibody-Astrocyte immunohistochemical observation found:The results of nerve morphology observation were basically similar to those seen in HE staining. Because of the specific binding of Anti-S100 antibody-Astrocyte with Schwann cells, the specific immune response of the cells showed the morphology of Schwann cells. The number of Schwann cells in Group El was the most, and the myelin sheath was the thickest, and the myelin sheath layer was clear and tidy. The number of Schwann cells in Group E2 was less than that in Group E1, and the myelin sheath was thinner, and the myelin sheath layer was not very clear and tidy. The number of Schwann cells in Group E3 was the least, and the myelin sheath was the thinnest, and the myelin sheath layer was obscure. The number of Schwann cells in N group was less, and the myelin sheath was thin, and the layer of myelin sheath was not obvious.Anti-200kD Neurofilament Heavy immunohistochemical observation found:The results of nerve morphology observation were basically similar to those seen in HE staining. Because of the specific binding of Anti-200kD Neurofilament Heavy with the neurofilament, the specific immune response of the cells showed the morphology of neurofilament of the axons. In Group E1, the regenerated nerve fibers were arranged in a neat and orderly manner, with a large number of nerve fibers, and the diameter of the regenerated nerve fibers was the thickest. In Group E2, the number of regenerated nerve fibers was less, and the neurofilaments were arranged less orderly, and the diameter of the regenerated nerve fibers was thinner. In Group E3, the regenerative nerve showed expansive growth, curled into a group, arranged in disorder, and the number of nerve regeneration was the least. In Group N, the regenerated nerve fibers is disordered, diameter of the nerve was thin. The measurement of the number of nerve fibers showed:Group E1> Group E2> Group N> Group E3, and the difference was statistically significant (P< 0.05).5.Ultrastructure of regenerative nerve observation:the distal lmm of regenerated nerves were separated and cut and fixed by glutaraldehyde, stained with uranyl acetate and lead citrate. Observation of ultrastructure of nerve regeneration was performed throgh transmission electron microscope.In Group E1, the axons is round in cross section, and the regenerated nerve fibers were arranged in a neat and orderly manner, with a large number of neurofilaments. The myelin sheath was most mature and the thickest, and the lamellae neatly wrapped around axons in concentric circles.In Group E2, the axons is basically round in cross section, partly twisted, and the regenerated nerve fibers were arranged in a less neat and orderly manner, with less neurofilaments. The myelin sheath was less mature and thinner, and the lamellae neatly wrapped around axons in concentric circles basically. In Group E3, the cross-sectional shape of the axons was obviously irregular. The regenerative nerve were arranged in disorder, and the number of nerve regeneration was the least, and the diameter of the smallest. The myelin sheath is the thinnest, and the myelin sheath is distorted and deformed. The lamellae were arranged disorderly, extremely loose and blurred. In Group N, the cross section shape of axons is irregular. The number of nerve regeneration was few, and the diameter is small. The myelin sheath is the thin, and the myelin sheath is partly distorted and deformed.6.Measurement of muscle wet weight ratio:At the 6th week after the operation, the muscle wet weight ratio of bilateral anterior lateral muscle groups of the lower leg in rats showed:Group E1> Group E2> Group N> Group E3, and the difference was statistically significant (P< 0.05).[Conclusion]When repairing the peripheral nerve injury, the small gap suture is better than the traditional gapless suture. The optimal length of the gap for nerve regeneration is around 1 mm. The longer the gap, the worse the regeneration. |
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